Probiotic bacteria from 10 different traditional Iranian cheeses: Isolation, characterization, and investigation of probiotic potential

Abstract In this study, 10 different traditional Iranian cheeses, which are still consumed by people in rural areas of Iran, were examined to isolate new strains of probiotic bacteria. Isolated bacteria were identified by 16s rRNA gene amplification and subjected to series of in vitro tests to find out their probiotic potential. A total of 2345 colonies were collected and 465 of them were confirmed as lactic acid bacteria (LAB), of which Lactiplantibacillus plantarum, Lactobacillus bulgaricus, and Lacticaseibacillus casei were the top three isolated bacteria. Among the different species of LAB isolated in this study, Lactip. plantarum was the most isolated species, and seven isolates had the significant criteria for being a probiotic strain than other isolates indicating the most adaptable properties of this species. Lactiplantibacillus plantarum was the most resistant bacteria in the bile resistance test and was also the most durable bacteria in gastrointestinal conditions, for example, acidic environment (pH = 2.5) and trypsin. In contrast, Lacticaseibacillus casei was the most susceptible bacterial strain. Lactobacillus rhamnosus showed the most antibacterial effect against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. This study showed that probiotic strains isolated from local cheeses could be considered as suitable biopreservatives and used as specific starter cultures for the production of functional cheeses.


| INTRODUC TI ON
Many advantages were reported for the consumption of probiotics (Alemohammad et al., 2022;Sharma et al., 2021). Beneficial effects of probiotics depend on their transport and survival through the gastrointestinal tract, which is also affected by initial numbers of probiotics in food, storage condition, food processing, food matrix, etc. (Behare et al., 2021). Among many foods that have been stud- ied for this purpose, cheese is the most interesting case to deliver these bacteria into the intestine (Karimi et al., 2011). This could be associated with the relatively high pH and fat content of cheese which exhibit a protective effect for the survival of probiotics in food and the gastrointestinal tract. Cheese and dairy products have been introduced among the fastest thriving food products in recent years worldwide (Elleuch et al., 2020). Many traditional dairy products and cheeses are produced and consumed in Iran, particularly rural regions. Traditional cheeses such as Lighvan (Ehsani et al., 2018), Koopeh cheese (Ehsani et al., 2018), and Shal fresh cheese could play a role in selecting new potential probiotic bacteria.
Many probiotics have been isolated from cheese, such as lactic acid bacteria (LAB) and Bifidobacteria with different strains. Among all LAB, Lactiplantibacillus plantarum, Lactic. casei, Limosilactobacillus fermentum, and Lactobacillus rhamnosus were more frequent (Ningtyas et al., 2019). LAB have been incorporated with different cheeses such as cheddar cheese, mascarpone cheese, and cottage cheese (Ningtyas et al., 2019). LAB strains are categorized as generally recognized as safe (GRAS) (Tulumoğlu et al., 2014), which makes them suitable for utilization in foods by researchers and industries.
Meanwhile, industries seek to produce new probiotic products in response to consumers' high demand.
One of the best approaches to find new probiotic strains is the investigation of traditional foods. These new strains can be used to produce new probiotic foods (Ehsani et al., 2018). To the best of our knowledge, traditional foods which are produced under nonindustrial conditions may contain different and unique bacterial compositions compared to industrial and modern foods (Gupta et al., 2021). Investigation of these traditional foods may lead to the isolation of novel probiotic strains with different properties (Adikari et al., 2021).
Overall, finding novel probiotic strains with high survival rates in food processing stages and high resistance to gastrointestinal juices due to mechanisms such as pH homeostasis, restriction of proton permeation, and enhancement of proton pumps is of great importance to observe their beneficial effects. Finding novel probiotic strains is also essential to provide diversity for probiotic foods and supplements as it has been revealed that diversity of gut microbiota is associated with health (Faintuch & Faintuch, 2019). In this study, we investigated 10 different traditional Iranian cheeses to isolate new strains of LAB. We also analyzed the identified bacteria to find out their resistance and survivability in the rough in vitro conditions of the gastrointestinal tract, as well as their antibacterial activity and their resistance to pathogenic bacteria.

| MATERIAL S AND ME THODS
2.1 | Traditional cheese sampling

| Isolation of bacteria from cheese samples
Isolation of bacteria was carried out according to Hassanzadazar et al. (2017) with slight modifications. Ninety-milliliter sterile solution of 0.85% sodium chloride (Sigma-Aldrich) was added to 10 g samples and shaken for 10 min with a shaker (Heidolph) to obtain suspensions.
Thirty milliliters of each sample were inoculated into Erlenmeyer flask with 300 ml de Man, Rogosa and Sharp (MRS) broth and incubated at the anaerobic conditions at 37°C for 24 h. Twenty-five milliliters of the latter was then centrifuged for 20 min at 10,000 g and sedimented mass cells were transferred to 10 ml phosphate buffer solution (pH = 2.5) and incubated at 37°C for 2 h. Subsequently, 0.1 ml sample was spread plated on MRS agar for 24-48 h at 37°C anaerobically and random colonies (flat or raised with grayish white color, smooth, rough, or intermediate) were selected for confirmation by some routine tests such as morphological evaluation by a microscope, Gram staining, and catalase test. Bacterial isolates characterization was performed by growth in MRS broth for 5 days at 30°C, 35°C, and 45°C, growth in MRS broth with 3%, 4%, and 6.5% NaCl at 30°C for 2 days, and gas production from glucose, fructose, sorbose, mannose, and xylose . Confirmed colonies were subcultured three times on MRS media for purification. Confirmed colonies were transferred into brain heart infusion broth (BHI) (Oxoid) supplemented with 15% (wt/vol) glycerol and stored at −80°C.

| DNA extraction, 16S rRNA gene amplification by PCR, and phylogenetic inference
DNA extraction was carried out according to the manufacturer's procedure (Qiagen DNA extraction kit) (Gharbi et al., 2019), and the purified DNA was used as a template for the PCR assay. For sequencing assay, the 1532 bp DNA fragment of 16s rRNA gene was targeted by using 5-AGAGTTTGATCCTGGCTCAG-3 and 5-CTACGGCTACCTTGTTACGA-3 primers (Denazist Asia) (Hou et al., 2018). The reaction mixture and PCR conditions for both PCRs were performed according to Scarpellini et al. (2002) using only one set of primers as a diagnostic PCR. Amplification reactions were carried out in thermocycler (Techne TC, 3000, England). The DNA was electrophoresed in 1% agarose gel containing ethidium bromide.
The PCR products (ca. 1500 bp) were purified and submitted for sequencing at the Macrogen (South Korea). At first, all sequences were trimmed at both ends and compared along with homologous sequences obtained from BLAST analysis (https:// blast.ncbi.nlm.nih.gov/Blast.cgi). All of them were aligned using Clustal W and finally were subjected to phylogenetic analysis.
Phylogenetic trees were constructed using MEGAX, and unweighted pair group method with arithmetic means (UPGMA) with 1000 replicates bootstrapping was employed. BioEdit Sequence Alignment Editor program (Hall, 1999) was used for trimming and aligning the sequences.

| Bile resistance
After the growth of each strain on MRS agar, it was transferred to sterile saline solution (0.85%) to make the 1.0 McFarland suspension. Of the suspension, 10 µl was spotted on the agar plates with 0.3%, 0.5%, 1.0%, and 2.0% (w/v) Ox-bile (Sigma-Aldrich) after 30 min, 1, 1.5, and 2 h, respectively. Plates were incubated anaerobically at 37°C and were evaluated after 24 h. Plates with no bacterial colony are considered negative, and the ones with the growth of colonies are considered positive. Plates with the absence of Ox-bile were considered as control.

| Simulation of gastrointestinal juices
The tolerance of bacteria was evaluated by the modified method of

| Determination of transient tolerance
Each strain was incubated in MRS broth at 37°C for 18 h, followed by centrifugation at 2000 g for 15 min. Overnight bacterial cell cultures (18 h) were collected, added to sterile normal saline (0.85% NaCl, w/v), and inoculated into simulated gastric juice with different pH (2.5, 3.0, and 4.0) or PBS solution (pH = 2.5, 3.0, and 4.0). Total viable count (TVC) was investigated for both simulated gastric juice and PBS solution at 0, 1, 2, 3, and 4 h at 37°C to assess the resistance to gastric juice.
To simulate the passage of bacteria through the gastrointestinal tract, after 3 h of incubation into the gastric juice (pH = 3.0), 1 ml of each strain was added to 9 ml of simulated intestinal juice with pH = 8.0 and incubated at 37°C. Resistance of bacteria to transit through the small intestine was measured at 37°C by TVC method for 0, 1, 2, 3, 4, 8, and 12 h (Hashemi et al., 2014).

| Antibiotic resistance
A single colony of LAB was selected, inoculated into a 10-ml tube containing Mueller-Hinton broth (Merck; CLSI, 2006), and incubated at 37°C. When turbidity of tube reached 0.5 McFarland, it was streak cultured on a plate containing 90% (w/v) Mueller-Hinton agar and 10% (w/v) MRS dehydrated broth (pH = 6.7). After implantation of the antibiotic disc on the plates, they were incubated at 37°C for 24-48 h.
Samples with a zone diameter of ≤15 mm are considered resistant. All antibiotic susceptibility tests were carried out in triplicate.

| Antibacterial activity of probiotic strains
Each strain was cultivated in 30 ml MRS broth at 37°C for 1 day and then centrifuged at 10,000 g for 15 min. A 0.22µm filter was used to omit remaining cells from the supernatant. NaOH 1 M was utilized to adjust the pH of 1 ml filtered supernatant (final pH 6.5-7

| Statistical analysis
Statistical analysis was carried out using SPSS software version 16 (SPSS Inc.) and Microsoft Excel.

| Isolation and identification of probiotic strains from traditional cheeses
A total of 2345 colonies were collected from all kinds of cheese samples, and from each morphological type, at least one colony was selected. Among all colonies, only 489 of them were Gram-positive and catalase-negative, and selected as suspected LAB.
A number of colonies collected from each type of cheese and the number of confirmed LAB in samples are shown in Figures 2 and 3 Milani et al. (2017) isolated LAB from Kurdish cheese during 60 days of ripening.
They reported that the predominant LAB were Lactip. plantarum which was consistent with the present study. This was also confirmed by Navidghasemizad et al. (2009), who demonstrated that Lactip. plantarum was predominant among LAB in Lighvan cheese.
Lactiplantibacillus plantarum showed most of the criteria for being a probiotic strain as many other studies. Its high adaptable properties are due to the enzymatic ability, carbohydrate metabolism, and owning a large genome (Morelli et al., 2004). According to the study of Milani et al. (2017), the microbial community of LAB was dynamic during ripening, and at the final phase, Lactip. plantarum and Lactobacillus paracasei were the predominant LAB. This dynamic can be affected by factors such as temperature, season, and the ability of bacteria to utilize nutrient resources which may lead to alteration of the predominant bacteria.

| Bile resistance
Ingested probiotic bacteria must survive the harsh conditions of the gastrointestinal tract and reach the large intestine to exert their beneficial effects. Herein, bacteria must push through several obstacles, such as bile salts. Therefore, in this study, the resistance of bacteria to several bile concentrations (0.3%, 0.5%, 1%, and 2%) were evaluated. The results are represented in Table 1

| Determination of resistance to simulated gastrointestinal juices
Other obstacles for probiotic bacteria are gastrointestinal juices such as pepsin and trypsin. The tolerance of LAB strains to gastric pepsin and trypsin is presented in Table 2. Within the first hour of treatment with pepsin at pH 2.5, all bacterial strains showed resistance, but Lactip. plantarum exhibited the best ad-  plantarum to tolerate the acidic environment. Hamon et al. (2014) stated that dTDP-glucose 4,6-dehydratase, 3-oxoacyl-synthase II, and dTDP-4-dehydrorhamnose 3,5-epimerase play an essential role in the biogenesis of cell envelope and grant the Lactip. plantarum durability in acidic conditions. In addition, high amounts of enzymes and amino acids and cell membrane integrity were proposed as influential factors to prevent bacterial death from acid stress (Guo et al., 2017).

| Antibiotic resistance test
Antibiotic resistance is one of the most important criteria for the selection of probiotic strains. Transferable genetic materials such as antibiotic resistance genes can be spread to pathogenic bacteria. The result of the antibiotic resistance test is depicted in Table 3. plantarum were resistant to gentamycin and streptomycin. In another study, 10 different types of traditional cheeses in Brazil were assessed for the isolation of LAB strains (Margalho et al., 2020). The results showed that streptomycin could not prevent the growth of LAB strains, while tetracycline was the most potent antibiotic against them, followed by erythromycin, ceftazidime, and penicillin. Other studies showed that St. thermophilus, Leu. mesenteroides (Ammor et al., 2007), and several LAB strains (Ouwehand et al., 2016) were resistant to streptomycin, gentamycin, and kanamycin. Hashemi TA B L E 1 Resistance of 15 identified lactic acid bacteria isolated from traditional cheeses to 0.3%, 0.5%, 1%, and 2% bile concentrations

Streptococcus thermophilus
53.3  Antimicrobial resistance of probiotic bacteria could be intrinsic, which might be ascribed to chromosomal mutation or acquiring plasmid from other bacteria. It should be noticed that antibiotic-resistant probiotics with adopted mobile genetic material should not be added to feed and food to prevent the spread of antibiotic resistance.

| Antimicrobial activity of probiotic strains
Probiotic strains isolated from traditional cheeses were screened for antimicrobial activity against five different microorganisms. The results are shown in can be found in extracellular secretions (Dasari et al., 2014).  In this study, screening all performed tests were reviewed, and finally seven promising isolates that had shown better results like resistance more than 1.5 h against bile salt concentration and better resistance to simulated gastrointestinal juices (pH = 2.5) for 4 h and 12 h were subjected for 16S rRNA sequencing. Results demonstrated agreement between the phenotypic and genotypic methods in recognizing isolates (MT1-MT6) except for MT3, which showed the most similarity to Lactoc. lactis ( Figure 1).

| CON CLUS ION
In this study, we investigated 10 traditional Iranian cheeses to isolate new probiotic strains. We also tested their adaptation to gut conditions, antimicrobial activity, resistance to pathogenic bacteria, and phylogenetic analysis. The data showed that among the different species of LAB isolated in this study, Lactip. plantarum was the most isolated species, and six isolates had the significant criteria for being a probiotic strain than other isolates indicating the most adaptable properties of this species. Also, this study showed the excellent potential of local cheeses of this region of Iran in terms of having probiotic strains, which can be considered as suitable biopreservatives and can be used as specific starter cultures for the production of functional cheeses. It should be taken into consideration that analysis of technological properties of isolated probiotics was one of the limitations of our study, and should be carried out in further studies. Isolated probiotic bacteria can be added to different foods and their desirable effects, such as antimicrobial activity, should be tested.

ACK N OWLED G EM ENTS
The authors express sincere appreciation to the Ahvaz Jundishapur University of Medical Sciences (grant number NRC-9801) and the Mashhad University of Medical Sciences (grant number 961217) for their financial support.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data available on request from the authors.